1. Field of the Invention
This invention is in the field of biochemistry and specifically relates to genetic toxicology.
2. Description of the Prior Art
The use of lower organisms to evaluate the genetic effects of chemical and physical agents began in the studies of mustard gas effects on Drosophilia during World War II, and rapidly progressed with the use of prokaryotes and neurospora. However, work with mammalian cells was hindered by lack of technical development until Puck and Marcus provided a cloning technique for the HeLa human cell line. Puck, T.T. and Marcus, P. I., Proc. Nat. Acad. Sci., 41, 432-437 (1955).
Szybalski and Syzbalska developed a forward and backward selective system for detecting mutants negative or positive for the activity of x-linked hypoxanthine guanine phosphoribosyl transferase (HGPRT) in a heteropoloid human line, D98. Szybalski, W. and Szybalska, E. H., "Drug Sensitivity as a Genetic Marker for Human Cell Lines," Univ. Mich. Med. Bull., 28, 277-93 (1962). However, they observed no mutagenic effects when this line was treated with a series of physical and chemical agents known to be effective mutagens in other biosystems. Szybalski, W., Cold Spring Harbor Symposium on Quantitative Biology, 29, 151-159 (1964).
Later work with mammalian cell mutagenesis centered on the use of two near-diploid hamster lines (CHO) and V79, for which the dose-dependent mutagenic effects of ultraviolet and ionizing radiation were studied, as well as a series of chemicals. Kao, F. T. and Puck, T. T., "Genetics of Somatic Mammalian Cells." IX. Quantitation of Mutagenesis by Physical and Chemical Agents, J. Cell Physiol., 74, 245-257 (1969); Chu, E. H. Y., P. Brimer, K. B., Jacobsen and E. V. Merriam, "Mammalian Cell Genetics. I. Selection and Characterization of Mutations Auxotrophic for L-glutamine or Resistant to 8-azaguanine in Chinese Hamster Cells in Vitro," Genetics, 62, 359-377 (1969); Arlett, C. F. and J. Potter, "Mutation to 8-azaguanine Resistance Induced by .gamma.-irradiation in a Chinese Hamster Cell Line," Mutation Res., 13, 59-65 (1971); Bridges, B. A. and J. Huckel, "Mutagenesis of Cultures Mammalian Cells by X-irradiation and Ultraviolet Light," Mutation Res., 10, 141-151 (1970).
More recent studies have attempted to extend the mutagenesis protocols originally established for cell lines of rodent origin to those of human origin. Albertini, R. J., and Demars, R., "Detection and Quantification of X-ray-induced Mutation in Cultured, Diploid Human Fibroblasts," Mutat. Res., 18, 199-244 (1973); Maher, V. M. and Wessel, J. E., "Mutations to Azaguanine Resistance Induced in Cultured Diploid Human Fibroblasts by the Carcinogen N-acetoxy-2-acetylaminofluorene," Mutat. Res., 28, 277-284 (1975). Researchers have noticed marked differences between human and rodent cells in their ability to repair chemically induced damage to DNA, and variations may be expected among other species. Buhl, S. N. and Regan, J. D., J. Biophys., 14, 519-527 (1974); and Rauth, A. M., Tammemagi, M., and Hunter, G., J. Biophys., 14, 209-220 (1974). Because of these differences, the use of human cells may prove necessary, and is certainly desirable, in obtaining relevant data for predicting the hazard that a particular chemical or other suspected mutagen poses for human beings.
One serious problem with most present protocols for performing mammalian cell mutation assays, however, is that the observed mutant fraction varies as a function of time after treatment with a selective agent. This finding is well documented for the appearance of 6-thioguanine (6TG) or 8-azaguanine (8AG)-resistant mutants in various rodent fibroblast systems and in human fibroblasts. Chu, E. H. Y. and Malling, H. V., Proc. Natl. Acad. Sci. U.S.A., 61, 1306-1312 (1968); Bridges, B. A. and Huckle, J., Mutat. Res., 10, 141-151 (1970); Arlett, C. F. and Harcourt, S. A., Mutat. Res., 14, 431-437 (1972); Maher, V. M. and Wessel, J. E., Mutat. Res., 28, 277-284 (1975). Recently, similar instability of a mutant fraction resistant to ouabain was reported for a Chinese hamster fibroblast line after methylnitronitrosoguanidine (MNNG) treatment. Davies, P. J. and Parry, J., Genet. Res., 24, 311-314 (1974). Such findings are, of course, inconsistent with the expected bahavior of stable hereditary changes.
One researcher has reported maintaining a mutagen-treated mouse cell population in near exponential growth for 2 weeks by frequent passaging to fresh plates. By adding a selective agent, 6-thioguanine, to the plates at various times after mutagen treatment and then measuring the fraction of recovered mutants, he has shown that the mutant fraction increases over a period of 8-10 days, finally reaching a stable maximum. Chasin, L., "The Effect of Ploidy on Chemical Mutagenesis in Cultured Chinese Hamster Cells," J. Cell Physiol., 82, 299-308 (1973). A second laboratory has reported confirmation of this finding with anchorage-dependent cells. Hsie, A. W., Brimer, P. A., Mitchell, T. J. and Goslee, D. G., "The Dose-response Relationship for Ethyl Methane Sulfonate-induced Mutations at the Hypoxanthine-guanine Phosphoribosyl Transferase Locus in Chinese Hamster Ovary Cells," Somatic Cell Genetics, 1, 247-261 (1975).
Additionally, some researchers have recently added mutagens to human lymphoblasts, but they failed to measure resulting mutagenesis in a quantitative way. Sato, K., Slesinski, R. S. and Littlefield, J. W., Proc. Natl. Acad. Sci. U.S.A., 69, 1244-1248 (1972).